The overall goal of this proposal is to elucidate the mechanisms by which key prostanoid synthetic enzymes, notably cyclooxygenase (COX-1 and COX-2), thromboxane A2 synthase (TXAS) and prostacyclin synthase (PGIS) are transcriptionally activated and regulated. These enzymes undergo autoinactivation during catalysis and, hence, have very short half-life. Transcriptional upregulation is a vital mechanism by which their cellular levels are maintained. Recently, novel observations have been made which will serve as basis for future experiments proposed in this application. The applicant postulates that salicylate blocks COX-2 induction by a cell cycle dependent mechanism related to the expression of a novel gene, CUL-3 cloned in their laboratory. They further postulate that the extent of TXA2 synthesis in monocyte-macrophages is controlled by coregulation of COX and TXAS expression and colocalization of these enzymes. To test these hypotheses, the applicant proposes five specific aims: (1) to determine positive feedback transcriptional regulation of COX-2 by prostaglandins and cAMP analogs, (2) to study the control of COX-2 induction by salicylate, (3) to evaluate the functional role of CUL-3 in cell cycle dependent suppression of COX-2 induction by salicylate, (4) to assess the influence of concurrent regulation of CCX and TXAS expressions in TXA2 biosynthesis, and (5) to elucidate the mechanism of TXAS transcription and regulation. The applicant will employ state-of-art biochemical, molecular-cellular, biological and molecular genetic approaches to unravel important mechanistic issues. The proposed work will provide new, important information on prostanoid synthetic enzymes. More importantly, novel concepts regarding the mechanism of action of salicylate, cell cycle control and transcriptional activation of housekeeping genes will be generated from these studies. Information derived from these studies will have great impact not only on understanding the fundamental biosynthesis of prostanoids but also on designing new drugs for controlling important diseases such as inflammation, tumor and cardiovascular diseases.